The present invention relates to a method for cloning restriction-modification systems, to the clones produced thereby, and to methods for producing restriction and/or modification enzymes from the clones. This invention also relates to clones for the DdeI and BamHI restriction endonucleases and modification methylases, and related methods for the production of these clones and enzymes.
Restriction endonucleases are a class of enzymes that occur naturally in bacteria. When they are purified away from other contaminating bacterial components, restriction endonucleases can be used in the laboratory to break DNA molecules into precise fragments. This property enables DNA molecules to be uniquely identified and to be fractionated into their constituent genes. Restriction endonucleases have proved to be indispensable tools in modern genetic research. They are the biochemical `scissors` by means of which genetic engineering and analysis is performed.
Restriction endonucleases act by recognizing and binding to particular sequences of nucleotides (the `recognition sequence`) along the DNA molecule. Once bound, they cleave the molecule within, or to one side of, the sequence. Different restriction endonucleases have affinity for different recognition sequences. Close to one hundred different restriction endonucleases have been identified among the many hundreds of bacterial species that have been examined to date.
Bacteria tend to possess at most only a small number restriction endonucleases per species. The endonucleases typically are named according to the bacteria from which they are derived. Thus, the species Haemophilus aegyptius, for example synthesizes 3 different restriction endonucleases, named Hae I, Hae II and Hae III. Those enzymes recognize and cleave the sequences (AT)GGCC(AT), PuGCGCPy and GGCC respectively. Escherichia coli RY13, on the other hand, synthesizes only one enzyme, EcoR I, which recognizes the sequence GAATTC.
In nature, restriction endonucleases play a protective role in the welfare of the bacterial cell. They enable bacteria to resist infection by foreign DNA molecules like viruses and plasmids that would otherwise destroy or parasitize them. They impart resistance by scanning the lengths of the infecting DNA molecule and cleaving them each time that the recognition sequence occurs. The break-up that takes place disables many of the infecting genes and renders the DNA susceptible to further degradation by non-specific endonucleases.
A second component of bacterial protective systems are the modification methylases. These enzymes are complementary to restriction endonucleases and they provide the means by which bacteria are able to identify their own DNA and distinguish it from foreign, infecting DNA. Modification methylases recognize and bind to the same nucleotide recognition sequence as the corresponding restriction endonuclease, but instead of breaking the DNA, they chemically modify one or other of the nucleotides within the sequence by the addition of a methyl group. Following this methylation, the recognition sequence is no longer bound or cleaved by the restriction endonuclease. The DNA of a bacterial cell is always fully modified, by virtue of its modification methylase, and it is therefore completely insensitive to the presence of the endogenous restriction endonuclease. It is only unmodified, and therefore identifiably foreign, DNA that is sensitive to restriction endonuclease recognition and attack.
With the advent of genetic engineering technology, it is now possible to clone genes and to produce the proteins and enzymes that they encode in greater quantities than are obtainable by conventional purification techniques. The key to isolating restriction endonuclease clones is to develop a simple and reliable method to identify such clones within complex `libraries`, i.e. populations of clones derived by `shotgun` procedures, when they occur at frequencies as low as 10.sup.-4 to 10.sup.-3. Preferably, the method should be selective, such that the unwanted majority of clones are destroyed while the rare desirable clones survive.
Type II restriction-modification systems are being cloned with increasing rapidity. The first cloned systems used bacteriophage infection as a means of selectively isolating restriction endonuclease clones (for PstI Walder et al., Proc. Nat. Acad. Sci. 74 1503-1507 (1981), HhaII Mann et al. Gene 3: 97-112 (1981). Since the presence of restriction-modification systems in bacteria enable them to resist infection by bacteriophages, cells that carry cloned restriction modification genes can in principle be selectively isolated as survivors from libraries that have been exposed to phage. This method has been found, however, to have only limited value. Specifically, it has been found that cloned restriction-modification genes do not always manifest sufficient phage resistance to confer selective survival. Another cloning approach involves transferring systems initially characterized as plasmid-borne into E. coli cloning plasmids (EcoRV, Bougueleret et al., Nucl. Acid. Res. 129:3659-3676 1984: PaeR7, Gingeras and Brooks, Proc. Natl. Acad. Sci. USA 80:402-406 1983, Theriault and Roy, 1982; PvuII, Blumenthal et al., J. Bacteriol. 164:501-509 1985. Finally, a growing number of systems are now being cloned by selection for an active methylase gene (BsuRI, Kiss et al., Nucl. Acid. Res. 13:6403-6421 1986; TaqI); since the two genes are often closely linked, both genes are cloned simultaneously. However, the methylase selection does not always yield a complete restriction system (BspRI, Szomolanyi et al., Gene 10:219-225 1980; MspI, Walder et al., J. Biol. Chem. 258:1235-1241 1983). Even attempts to clone larger regions adjacent to the methylase gene can fail to produce an active endonuclease gene.
In some systems the cloning problem may lie in trying to introduce the endonuclease gene into a host not adequately protected by methylation. If the methylase gene and endonuclease gene are introduced on a common DNA fragment, the former gene product must modify the host before the latter gene product cleaves the host genome.
Another obstacle to cloning these systems in E. coli was discovered in the process of cloning diverse methylases. Many E. coli strains (including those normally used in cloning) have systems that block introduction of DNA containing heterologous cytosine methylation into the cells (Raleigh and Wilson, submitted for publication). Therefore, it is also necessary to carefully consider which E. coli strain(s) to use for cloning.
Because purified restriction endonucleases, and to a lesser extent, modification methylases, are useful tools for characterizing and rearranging DNA in the laboratory, there is a commercial incentive to develop methods of obtaining strains of bacteria that synthesize these enzymes in abundance. Such strains would be useful because they would simplify the task of purification as well as providing the means for production in commercially useful amounts.